Differential Effects of Natriuretic Peptide Stimulation on Tissue-Engineered Cartilage STEPHEN D. WALDMAN, Ph.D., 1,2 YASMINE USMANI, B.Sc., 3 M. YAT TSE, Ph.D., 3 and STEPHEN C. PANG, Ph.D. 2,3 ABSTRACT Tissue engineering is a promising approach for articular cartilage repair; however, it still has proven a challenge to produce substantial quantities of tissue from the limited number of cells that can be extracted from a single individual. Although several approaches have been investigated to enhance the production of cartilaginous tissue in vitro, relatively few techniques exist to reliably increase the population of cells needed for this approach. Alternatively, a single modulator of chondrocyte function, such as the C-type natriuretic peptide (CNP), may serve to address both of these issues. CNP is expressed in the growth plate and regulates cartilage growth through chondrocyte proliferation and differentiation. Thus, the purpose of this study was to determine the effects of CNP stimulation on tissue-engineered cartilage. Isolated bovine articular chondrocytes were seeded on Millicell TM filters and cultured in the presence of CNP (10 pM to 10 nM) for 4 weeks. Stimulation with CNP resulted in differential effects depending on the dose of the peptide. Low doses of CNP (10 to 100 pM) elicited chondrocyte proliferation with a maximal response observed at 100 pM (43% increase in cellularity). However, high doses of CNP (10 nM) stimulated matrix deposition (36% and 137% increase in proteoglycans and collagen) without an associated change in tissue cellularity. CNP stimulation also downregulated the expression of type X collagen, an early hypertrophic marker associated with endochondral ossification. Thus, by regulating the dose of CNP, it may be possible to produce engineered tissue from the limited number of cells that can be reasonably extracted from a single individual for therapeutic purposes. INTRODUCTION R ESURFACING DAMAGED JOINT CARTILAGE with tissue formed in vitro is a promising new approach for ar- ticular cartilage repair. Isolated cartilage cells (chondro- cytes) grown in three-dimensional culture tend to maintain their phenotype and are capable of synthesizing a carti- laginous extracellular matrix (ECM) that is similar to that of the native tissue. 1–4 Although much progress has been made in refining this approach, it still has proven chal- lenging to produce enough tissue suitable for the repair of clinically relevant–sized cartilage defects from the limited number of cells that can be reasonably extracted from a single individual. Several approaches have been investigated to enhance the production of cartilaginous tissue in vitro. Techniques such as gene therapy, 5,6 stimulation by growth factors, 7,8 and mechanical conditioning 2,9,10 all have been shown to be effective at accelerating the production of cartilaginous ECM. Alternatively, however, cell sourcing still remains a major technical hurdle as there are relatively few methods available to reliably increase the population of cells needed 1 Department of Chemical Engineering and Department of Mechanical and Materials Engineering, Queen’s University, Kingston, Ontario, Canada. 2 Human Mobility Research Centre, Kingston General Hospital, Kingston, Ontario, Canada. 3 Department of Anatomy and Cell Biology, Queen’s University, Kingston, Ontario, Canada. TISSUE ENGINEERING: Part A Volume 14, Number 3, 2008 # Mary Ann Liebert, Inc. DOI: 10.1089/tea.2007.0035 441